Identification of key ancestors of modern germplasm in a breeding program of maize.

KEY MESSAGE: Probabilities of gene origin computed from the genomic kinships matrix can accurately identify key ancestors of modern germplasms Identifying the key ancestors of modern plant breeding populations can provide valuable insights into the history of a breeding program and provide reference genomes for next generation whole genome sequencing. In an animal breeding context, a method was developed that employs probabilities of gene origin, computed from the pedigree-based additive kinship matrix, for identifying key ancestors. Because reliable and complete pedigree information is often not available in plant breeding, we replaced the additive kinship matrix with the genomic kinship matrix. As a proof-of-concept, we applied this approach to simulated data sets with known ancestries. The relative contribution of the ancestral lines to later generations could be determined with high accuracy, with and without selection. Our method was subsequently used for identifying the key ancestors of the modern Dent germplasm of the public maize breeding program of the University of Hohenheim. We found that the modern germplasm can be traced back to six or seven key ancestors, with one or two of them having a disproportionately large contribution. These results largely corroborated conjectures based on early records of the breeding program. We conclude that probabilities of gene origin computed from the genomic kinships matrix can be used for identifying key ancestors in breeding programs and estimating the proportion of genes contributed by them.

Dent and Flint maize diversity panels reveal important genetic potential for increasing biomass production.

KEY MESSAGE: Genetic and phenotypic analysis of two complementary maize panels revealed an important variation for biomass yield. Flowering and biomass QTL were discovered by association mapping in both panels. The high whole plant biomass productivity of maize makes it a potential source of energy in animal feeding and biofuel production. The variability and the genetic determinism of traits related to biomass are poorly known. We analyzed two highly diverse panels of Dent and Flint lines representing complementary heterotic groups for Northern Europe. They were genotyped with the 50 k SNP-array and phenotyped as hybrids (crossed to a tester of the complementary pool) in a western European field trial network for traits related to flowering time, plant height, and biomass. The molecular information revealed to be a powerful tool for discovering different levels of structure and relatedness in both panels. This study revealed important variation and potential genetic progress for biomass production, even at constant precocity. Association mapping was run by combining genotypes and phenotypes in a mixed model with a random polygenic effect. This permitted the detection of significant associations, confirming height and flowering time quantitative trait loci (QTL) found in literature. Biomass yield QTL were detected in both panels but were unstable across the environments. Alternative kinship estimator only based on markers unlinked to the tested SNP increased the number of significant associations by around 40% with a satisfying control of the false positive rate. This study gave insights into the variability and the genetic architectures of biomass-related traits in Flint and Dent lines and suggests important potential of these two pools for breeding high biomass yielding hybrid varieties.

Fine mapping of qhir1 influencing in vivo haploid induction in maize.

Production of haploids by the in vivo haploid induction method has now become routine for generating new inbred lines in maize. In previous studies, a major quantitative trait locus (QTL) (qhir1) located in bin 1.04 was detected, explaining up to 66 % of the genotypic variance for haploid induction rate (HIR). Our objectives were to (1) fine-map qhir1 and (2) identify closely linked markers useful for marker-assisted breeding of new inducers. For this purpose, we screened a mapping population of 14,375 F2 plants produced from a cross between haploid inducer UH400 and non-inducer line 1680 to identify recombinants. Based on sequence information from the B73 reference genome, markers polymorphic between the two parents were developed to conduct fine mapping with these recombinants. A progeny test mapping strategy was applied to accurately determine the HIR of the 14 recombinants identified. Furthermore, F3 progeny of recombinant F2 plants were genotyped and in parallel evaluated for HIR. We corroborated earlier studies in that qhir1 has both a significantly positive effect on HIR but also a strong selective disadvantage, as indicated by significant segregation distortion. Altogether, we were able to narrow down the qhir1 locus to a 243 kb region flanked by markers X291 and X263.

Optimum design of family structure and allocation of resources in association mapping with lines from multiple crosses.

Family mapping is based on multiple segregating families and is becoming increasingly popular because of its advantages over population mapping. Athough much progress has been made recently, the optimum design and allocation of resources for family mapping remains unclear. Here, we addressed these issues using a simulation study, resample model averaging and cross-validation approaches. Our results show that in family mapping, the predictive power and the accuracy of quatitative trait loci (QTL) detection depend greatly on the population size and phenotyping intensity. With small population sizes or few test environments, QTL results become unreliable and are hampered by a large bias in the estimation of the proportion of genotypic variance explained by the detected QTL. In addition, we observed that even though good results can be achieved with low marker densities, no plateau is reached with our full marker complement. This suggests that higher quality results could be achieved with greater marker densities or sequence data, which will be available in the near future for many species.

Molecular marker assisted broadening of the Central European heterotic groups in rye with Eastern European germplasm.

Broadening the genetic base of heterotic pools is a key to ensure continued genetic gains in hybrid breeding and extend hybrid cultivation to new areas. In the present study, two Central European heterotic pools (Carsten and Petkus) and five Eastern European open-pollinated varieties (OPVs, Pop-1 to Pop-5) were studied with the objectives to (1) investigate the genetic diversity in OPVs and the heterotic pools using molecular and field data, (2) evaluate the molecular diversity among OPVs, (3) examine the combining ability for grain yield of the OPVs when crossed with testers in field trials, and (4) develop a strategy for targeted introgression of OPV germplasm into the heterotic pools. In total, 610 S(0) plants, 347 from OPVs and 263 from heterotic pools, were developed. Clones of the S(0) plants of OPVs were crossed with two testers belonging to each heterotic pool, while clones of heterotic pools were crossed with only the opposite tester. Testcrosses were evaluated for grain yield in multi-location trials. In addition, 589 S(0) plants were fingerprinted with 30 SSR markers. The data revealed that the Carsten pool has a narrow genetic base and should be the primary target for broadening the established heterotic pattern. Mean and genetic variance suggested that Pop-2 and Pop-4 are good candidates for introgression in Petkus pool and Pop-5 in Carsten pool. Nevertheless, introgression of Pop-5 in Carsten could reduce the genetic diversity between heterotic pools. Therefore, we suggest that either selected plants of Pop-5 should be introgressed or more Eastern European germplasm should be fingerprinted and field evaluated to identify promising germplasm for broadening the established heterotic pattern.

Genetic expectations of quantitative trait loci main and interaction effects obtained with the triple testcross design and their relevance for the analysis of heterosis.

Interpretation of experimental results from quantitative trait loci (QTL) mapping studies on the predominant type of gene action can be severely affected by the choice of statistical model, experimental design, and provision of epistasis. In this study, we derive quantitative genetic expectations of (i) QTL effects obtained from one-dimensional genome scans with the triple testcross (TTC) design and (ii) pairwise interactions between marker loci using two-way analyses of variance (ANOVA) under the F(2)- and the F(infinity)-metric model. The theoretical results show that genetic expectations of QTL effects estimated with the TTC design are complex, comprising both main and epistatic effects, and that genetic expectations of two-way marker interactions are not straightforward extensions of effects estimated in one-dimensional scans. We also demonstrate that the TTC design can partially overcome the limitations of the design III in separating QTL main effects and their epistatic interactions in the analysis of heterosis and that dominance x additive epistatic interactions of individual QTL with the genetic background can be estimated with a one-dimensional genome scan. Furthermore, we present genetic expectations of variance components for the analysis of TTC progeny tested in a split-plot design, assuming digenic epistasis and arbitrary linkage.

Impact of interpopulation divergence on additive and dominance variance in hybrid populations.

We present a theoretical proof that the ratio of the dominance vs. the additive variance decreases with increasing genetic divergence between two populations. While the dominance variance is the major component of the variance due to specific combining ability (sigma(SCA)(2)), the additive variance is the major component of the variance due to general combining ability (sigma(GCA)(2)). Therefore, we conclude that interpopulation improvement becomes more efficient with divergent than with genetically similar heterotic groups, because performance of superior hybrids can be predicted on the basis of general combining ability effects.

The role of epistasis in the manifestation of heterosis: a systems-oriented approach.

Heterosis is widely used in breeding, but the genetic basis of this biological phenomenon has not been elucidated. We postulate that additive and dominance genetic effects as well as two-locus interactions estimated in classical QTL analyses are not sufficient for quantifying the contributions of QTL to heterosis. A general theoretical framework for determining the contributions of different types of genetic effects to heterosis was developed. Additive x additive epistatic interactions of individual loci with the entire genetic background were identified as a major component of midparent heterosis. On the basis of these findings we defined a new type of heterotic effect denoted as augmented dominance effect di* that comprises the dominance effect at each QTL minus half the sum of additive x additive interactions with all other QTL. We demonstrate that genotypic expectations of QTL effects obtained from analyses with the design III using testcrosses of recombinant inbred lines and composite-interval mapping precisely equal genotypic expectations of midparent heterosis, thus identifying genomic regions relevant for expression of heterosis. The theory for QTL mapping of multiple traits is extended to the simultaneous mapping of newly defined genetic effects to improve the power of QTL detection and distinguish between dominance and overdominance.

Chilling tolerance of Central European maize lines and their factorial crosses.

BACKGROUND AND AIMS: Chilling-stress tolerance is a prerequisite for maize production under cool climatic conditions. The main goal of this study was to evaluate the Central European dent and flint pools for chilling tolerance during heterotrophic and early autotrophic growth in field trials and growth chamber experiments. METHODS: Five European flint and five dent inbreds and their 25 factorial crosses were evaluated in six natural environments, where chilling occurred, for chlorophyll concentration and plant height at the three-leaf stage, and plant height and fresh weight at the seven-leaf stage. In growth chambers, leaf 3 growth was analysed under cold and control conditions. KEY RESULTS: Comparing the field and growth chamber data, the strongest association was found between leaf elongation rate during cold nights and plant height at the three-leaf stage, with a weaker association with the seven-leaf stage. In the field, moderate correlations were observed between plant height at the three-leaf stage, and plant height and fresh weight at the seven-leaf stage, respectively. Furthermore, mid-parent and hybrid performance were only moderately correlated. CONCLUSIONS: The results suggest that heterotrophic and early autotrophic growth stages are controlled by different genetic factors or that maternal effects play a role. In addition, the findings showed that mid-parent performance is a poor predictor of hybrid performance. Consequently, test cross performance should be the target in quantitiative trait locus (QTL) mapping studies with the final goal of establishing marker-assisted breeding programmes for chilling-tolerant hybrids.

The length of the intact donor chromosome segment around a target gene in marker-assisted backcrossing.

Recurrent backcrossing is an established procedure to transfer target genes from a donor into the genetic background of a recipient genotype. By assessing the parental origin of alleles at markers flanking the target locus one can select individuals with a short intact donor chromosome segment around the target gene and thus reduce the linkage drag. We investigated the probability distribution of the length of the intact donor chromosome segment around the target gene in recurrent backcrossing with selection for heterozygosity at the target locus and homozygosity for the recurrent parent allele at flanking markers for a diploid species. Assuming no interference in crossover formation, we derived the cumulative density function, probability density function, expected value, and variance of the length of the intact chromosome segment for the following cases: (1) backcross generations prior to detection of a recombinant individual between the target gene and the flanking marker; (2) the backcross generation in which for the first time a recombinant individual is detected, which is selected for further backcrossing; and (3) subsequent backcross generations after selection of a recombinant. Examples are given of how these results can be applied to investigate the efficiency of marker-assisted backcrossing for reducing the length of the intact donor chromosome segment around the target gene under various situations relevant in breeding and genetic research.

Quantitative trait locus (QTL) mapping using different testers and independent population samples in maize reveals low power of QTL detection and large bias in estimates of QTL effects.

The efficiency of marker-assisted selection (MAS) depends on the power of quantitative trait locus (QTL) detection and unbiased estimation of QTL effects. Two independent samples N = 344 and 107 of F2 plants were genotyped for 89 RFLP markers. For each sample, testcross (TC) progenies of the corresponding F3 lines with two testers were evaluated in four environments. QTL for grain yield and other agronomically important traits were mapped in both samples. QTL effects were estimated from the same data as used for detection and mapping of QTL (calibration) and, based on QTL positions from calibration, from the second, independent sample (validation). For all traits and both testers we detected a total of 107 QTL with N = 344, and 39 QTL with N = 107, of which only 20 were in common. Consistency of QTL effects across testers was in agreement with corresponding genotypic correlations between the two TC series. Most QTL displayed no significant QTL x environment nor epistatic interactions. Estimates of the proportion of the phenotypic and genetic variance explained by QTL were considerably reduced when derived from the independent validation sample as opposed to estimates from the calibration sample. We conclude that, unless QTL effects are estimated from an independent sample, they can be inflated, resulting in an overly optimistic assessment of the efficiency of MAS.

Comparative Quantitative Trait Loci Mapping of Partial Resistance to Puccinia sorghi Across Four Populations of European Flint Maize.

ABSTRACT We mapped and characterized quantitative trait loci (QTL) for partial resistance to Puccinia sorghi and investigated consistency across different European flint maize populations. Four independent populations, containing 280 F(3) lines (AxB(I)), 120 F(5) lines (AxB(II)), 131 F(4) lines (AxC), and 133 F(4) lines (CxD) were produced from four European elite flint inbreds (A, B, C, and D) and genotyped at 89, 151, 104, and 122 restriction fragment length polymorphism marker loci, respectively. All F(n) lines were evaluated in field trials with two replications in three or five (AxB(I)) environments. Genotypic variance was highly significant for rust ratings in all populations, and heritabilities exceeded 0.64. Between 4 and 13 QTL were detected in individual populations using composite interval mapping, explaining between 33 and 71% of the phenotypic variance. Twenty QTL were distributed over all ten chromosomes, without preference to chromosomes 3, 4, 6, and 10, which harbor qualitatively acting Rp loci. In most cases, gene action was additive or partially dominant. Four pairs of QTL displayed significant digenic epistatic interactions, and QTL-environment interactions were observed frequently. Approximately half of the QTL were consistent between AxB(I) and AxB(II) or AxC and CxD; fewer were consistent between AxB(I) and AxC or CxD. In European flint maize germ plasm, conventional selection for partial rust resistance seems to be more promising than marker-assisted selection.

Quantitative trait Loci mapping of resistance to sugarcane mosaic virus in maize.

ABSTRACT Sugarcane mosaic virus (SCMV) is an important virus disease of maize (Zea mays) in Europe. In this study, we mapped and characterized quantitative trait loci (QTL) affecting resistance to SCMV in a maize population consisting of 219 F(3) or immortalized F(2) families from the cross of two European maize inbreds, D32 (resistant) x D145 (susceptible). Resistance was evaluated in replicated field trials across two environments under artificial inoculation. The method of composite interval mapping was employed for QTL detection with a linkage map based on 87 restriction fragment length polymorphism and 7 mapped microsatellite markers. Genotypic and genotype x environment interaction variances for SCMV resistance were highly significant in the population. Heritabilities ranged from 0.77 to 0.94 for disease scores recorded on seven consecutive dates. Five QTL for SCMV resistance were identified on chromosomes 1, 3, 5, 6, and 10 in the joint analyses. Two major QTL on chromosomes 3 and 6 were detected consistently in both environments. Significant epistatic effects were found among some of these QTL. A simultaneous fit with all QTL in the joint analyses explained between 70 and 77% of the phenotypic variance observed at various stages of plant development. Resistance to SCMV was correlated with plant height and days to anthesis.

Species-Specific Detection of the Maize Pathogens Sporisorium reiliana and Ustilago maydis by Dot Blot Hybridization and PCR-Based Assays.

Head smut of maize, caused by Sporisorium reiliana, may substantially reduce grain yield. The objective of the present study was to develop a highly specific and sensitive DNA-based assay for detection of S. reiliana and its differentiation from Ustilago maydis, a maize fungus inducing the symptomatically similar common smut disease. Plasmid libraries of S. reiliana and U. maydis were constructed using a shotgun cloning procedure. Clones containing strongly hybridizing species-specific DNA were selected by screening libraries with their own labeled genomic DNA, followed by cross-hybridization with genomic DNA of maize and other maize-pathogenic fungi. The selected clones were used to generate subclones with short insert fragments to facilitate PCR amplification for labeling and primer design for a PCR assay. Using Dig-dUTP labeled inserts, detection of less than 0.16 ng of fungal DNA was possible by dot blot hybridization. Sequences of insert fragments were determined to design primer pairs for a PCR-based assay. Primer pairs SR1 and SR3 are species-specific for S. reiliana, and UM11 is species-specific for U. maydis. The PCR-based assays can detect fungal DNA of less than 1.6 pg using SR1 and SR3, and 8 pg using UM11, irrespective of the presence of maize DNA. Use of SR1 and SR3 allowed detection of S. reiliana in the extracts of pith, node, and shank from S. reiliana-infected plants, but not in leaves. Thus, both the dot blot hybridization and the PCR-based assays provide a highly sensitive and reliable tool for detection and differentiation of corn smut caused either by S. reiliana or by U. maydis.

An incomplete enumeration algorithm for an exact test of Hardy-Weinberg proportions with multiple alleles.

Testing of Hardy-Weinberg proportions (HWP) with asymptotic goodness-of-fit tests is problematic when the contingency table of observed genotype counts has sparse cells or the sample size is low, and exact procedures are to be preferred. Exact p-values can be (1) calculated via computational demanding enumeration methods or (2) approximated via simulation methods. Our objective was to develop a new algorithm for exact tests of HWP with multiple alleles on the basis of conditional probabilities of genotype arrays, which is faster than existing algorithms. We derived an algorithm for calculating the exact permutation significance value without enumerating all genotype arrays having the same allele counts as the observed one. The algorithm can be used for testing HWP by (1) summation of the conditional probabilities of occurrence of genotype arrays with smaller probability than the observed one, and (2) comparison of the sum with a nominal Type I error rate alpha. Application to published experimental data from seven maize populations showed that the exact test is computationally feasible and reduces the number of enumerated genotype count matrices about 30% compared with previously published algorithms.

Temporal changes in allele frequencies in two European F(2) flint maize populations under modified recurrent full-sib selection.

Selection and random genetic drift are the two main forces affecting the selection response of recurrent selection (RS) programs by changes in allele frequencies. Therefore, detailed knowledge on allele frequency changes attributable to these forces is of fundamental importance for assessing RS programs. The objectives of our study were to (1) estimate the number, position, and genetic effect of quantitative trait loci (QTL) for selection index and its components in the base populations, (2) determine changes in allele frequencies of QTL regions due to the effects of random genetic drift and selection, and (3) predict allele frequency changes by using QTL results and compare these predictions with observed values. We performed QTL analyses, based on restriction fragment length polymorphisms (RFLPs) and simple sequence repeats (SSRs), in 274 F(2:3) lines of cross KW1265 x D146 (A x B) and 133 F(3:4) lines of cross D145 x KW1292 (C x D) originating from two European flint maize populations. Four (A x B) and seven (C x D) cycles of RS were analyzed with SSRs for significant allele frequency changes due to selection. Several QTL regions for selection index were detected with simple and composite interval mapping. In some of them, flanking markers showed a significant allele frequency change after the first and the final selection cycles. The correlation between observed and predicted allele frequencies was significant only in A x B. We attribute these observations mainly to (1) the high dependence of the power of QTL detection on the population size and (2) the occurrence of undetectable QTL in repulsion phase. Assessment of allele frequency changes in RS programs can be used to detect marker alleles linked to QTL regions under selection pressure.

Linkage disequilibrium in two European F(2) flint maize populations under modified recurrent full-sib selection.

According to quantitative genetic theory, linkage disequilibrium (LD) can hamper the short- and long-term selection response in recurrent selection (RS) programs. We analyzed LD in two European flint maize populations, KW1265 x D146 (A x B) and D145 x KW1292 (C x D), under modified recurrent full-sib selection. Our objectives were to investigate (1) the decay of initial parental LD present in F(2) populations by three generations of intermating, (2) the generation of new LD in four (A x B) and seven (C x D) selection cycles, and (3) the relationship between LD changes and estimates of the additive genetic variance. We analyzed the F(2) and the intermated populations as well as all selection cycles with 104 (A x B) and 101 (C x D) simple sequence repeat (SSR) markers with a uniform coverage of the entire maize genome. The LD coefficient D and the composite LD measure Delta were estimated and significance tests for LD were performed. LD was reduced by intermating as expected from theory. A directional generation of negative LD between favorable alleles could not be observed during the selection cycles. However, considerable undirectional changes in D were observed, which we attributed to genetic sampling due to the finite population size used for recombination. Consequently, a long-term reduction of the additive genetic variance due to negative LD was not observed. Our experimental results support the hypothesis that in practical RS programs with maize, LD generated by selection is not a limiting factor for obtaining a high selection response.

Trends in population parameters and best linear unbiased prediction of progeny performance in a European F(2) maize population under modified recurrent full-sib selection.

Recurrent selection is a cyclic breeding procedure designed to improve the mean of a population for the trait(s) under selection. Starting from an F(2) population of European flint maize (Zea mays L.) intermated for three generations, we conducted seven cycles of a modified recurrent full-sib (FS) selection scheme. The objectives of our study were to (1) monitor trends across selection cycles in the estimates of the population mean, additive and dominance variances, (2) compare predicted and realized selection responses, and (3) investigate the usefulness of best linear unbiased prediction (BLUP) of progeny performance under the recurrent FS selection scheme applied. Recurrent FS selection was conducted at three locations using a selection rate of 25% for a selection index, based on grain yield and grain moisture. Recombination was performed according to a pseudo-factorial mating scheme, where the selected FS families were divided into an upper-ranking group of parents mated to the lower-ranking group. Variance components were estimated with restricted maximum likelihood. Average grain yield increased 9.1% per cycle, average grain moisture decreased 1.1% per cycle, and the selection index increased 11.2% per cycle. For the three traits we observed, no significant changes in additive and dominance variances occurred, suggesting future selection response at or near current rates of progress. Predictions of FS family performance in Cn+1 based on mean performance of parental FS families in Cn were of equal or higher precision as those based on the mean additive genetic BLUP of their parents, and corresponding correlations were of moderate size only for grain moisture. The significant increase in grain yield combined with the decrease in grain moisture suggest that the F(2) source population with use of a pseudo-factorial mating scheme is an appealing alternative to other types of source materials and random mating schemes commonly used in recurrent selection.

Genetic drift and selection effects of modified recurrent full-sib selection programs in two F2 populations of European flint maize.

Selection response of a modified recurrent full-sib (FS) selection scheme conducted in two European flint F(2) maize (Zea mays L.) populations was re-evaluated. Our objectives were to (1) determine the selection response for per se and testcross performance in both populations and (2) separate genetic effects due to selection from those due to random genetic drift. Modified recurrent FS selection was conducted at three locations using an effective population size N(e) = 32 and a selection rate of 25% for a selection index, based on grain yield and grain moisture. Recombination was performed according to a pseudo-factorial mating scheme. Selection response was assessed using a population diallel including the source population and advanced selection cycles, as well as testcrosses with unrelated inbred line testers and the parental F(1) generation. Selection response per cycle was significant for grain yield and grain moisture in both populations. Effects of random genetic drift caused only a small reduction in the selection response. No significant selection response was observed for testcrosses, suggesting that for heterotic traits, such as grain yield, a high frequency of favorable alleles in the elite tester masked the effects of genes segregating in the populations. We conclude that our modified recurrent FS selection is an alternative to other commonly applied intrapopulation recurrent selection schemes, and some of its features may also be useful for increasing the efficiency of interpopulation recurrent selection programs.

Comparison of linkage maps from F2 and three times intermated generations in two populations of European flint maize (Zea mays L.).

Intermated mapping populations are expected to result in high mapping resolution for tightly linked loci. The objectives of our study were to (1) investigate the consequences of constructing linkage maps from intermated populations using mapping methods developed for F(2) populations, (2) compare linkage maps constructed from intermated populations (F(2)Syn3) with maps generated from corresponding F(2) and F(3) base populations, and (3) investigate the advantages of intermated mapping populations for applications in plant breeding programs. We constructed linkage maps for two European flint maize populations (A x B, C x D) by mapping 105 SSR markers in generations F(2) and F(2)Syn3 of population A x B, and 102 SSR markers in generations F(3) and F(2)Syn3 of population C x D. Maps for F(2)Syn3 were constructed with mapping methods for F(2) populations (Map A) as well as with those specifically developed for intermated populations (Map B). Both methods relate map distances to recombination frequencies in a single meiosis and, therefore, did not show a map expansion in F(2)Syn3 compared with maps constructed from the respective F(2) or F(3) base populations. Map A and B differed considerably, presumably because of theoretical shortcomings of Map A. Since loosely linked markers could not unambiguously be mapped in the F(2)Syn3 populations, they may hamper the construction of linkage maps from intermated populations.